The worldwide animal feed market, which includes livestock, poultry, aquaculture and pets is 475 million metric tons. In the United States 180 million metric tons are consumed, with corn (Zea mays L.) accounting for about 67% and soybean (Glycine max L.) meal for about 10% of the total. Corn and soybean products are also a major element of international trade.
Human food and animal feed derived from many grains are deficient in some of the ten essential amino acids (cysteine, isoleucine, lysine, methionine, phenylalanine, threonine, tyrosine, and valine) which are required in animal diets. In corn, lysine is the most limiting amino acid followed by tryptophan and the sulfur amino acids, methionine and cysteine, for the dietary requirements of many animals. The usefulness of soybean meal, which is rich in lysine and tryptophan, to supplement corn in animal feed is limited by the low sulfur amino acid content of the legume. When soybean meal is used to supplement the lysine levels of corn, the low levels of methionine in soybeans cause the blended feed to have an even lower level of methionine than the original corn. As a result, feed blends of corn and soybean typically still include methionine as an additive. A typical composition of chicken starter rations is shown in Table 1 [Powell et al., (1976) Poult. Science. 55:502-509].
TABLE 1 ______________________________________ Composition of Practical Chicken Starter Rations ______________________________________ Yellow Corn 57.25% Soybean Meal (49% protein) 29.00 Fish solubles 0.65 Wheat middlings 2.50 Delactosed whey 1.50 Costal bermudagrass, dehydrated 5.00 Minerals 0.25 Vitamins 0.25 Animal fat 0.25 DL-Methionine 0.10 Choline chloride 0.10 ______________________________________
Thus, a mechanism to increase the levels of particular amino acids within the plant seed for a given crop and a specific end use would eliminate the need to supplement mixed or single grain feeds with purified amino acids. Furthermore, the methionine requirements of poultry and swine (the two largest consumers of soybean meal accounting for 78% of the soy protein used in feeds [Wilcox, (1987) Agronomy 16:823]) decrease with age of the animal [Ensminger et al., (1978) Feeds and Nutrition, The Ensminger Publishing Co. Clovis, Calif.]. The ability to improve the essential amino acid content of soybean or corn in a controllable manner is therefore, extremely desirable. A solution to this problem is the design of a class of synthetic proteins which can be tailored to complement the deficiencies of any crop for use in feeding any animal of any age.
The amino acid content of seeds is determined primarily by the storage proteins which are synthesized during seed development and which serve as a major nutrient reserve following germination. The quantity of protein in seeds varies from about 10% of the dry weight in cereals to 20-40% of the dry weight of legumes. In many seeds the storage proteins account for 50% or more of the total protein. Because of their abundance, plant seed storage proteins were among the first plant proteins to be isolated. Only recently, however, have the amino acid sequences of some of these proteins been determined with the use of molecular genetic techniques. These techniques have also provided information about the genetic signals that control the seed-specific expression and the intracellular targetting of these proteins.
Although no plant seed storage proteins enriched in lysine relative to average lysine content of plant proteins have been identified, a number of sulfur-rich plant seed storage proteins have been identified and their corresponding genes isolated. A gene in corn for a 15 kD zein protein containing 11% methionine and 5% cysteine [Pedersen et al., (1986) J. Biol. Chem. 261:6279-6284] and a gene for a 10 kD zein protein containing 23% methionine and 3% cysteine have been isolated [Kirihara et al., (1988) Mol. Gen. Genet. 21:477-484; Kirihara et al., (1988) Gene 71:359-370]. Two genes from pea for seed albumins containing 8% and 16% cysteine have been isolated [Higgins et al., (1986) J. Biol. Chem. 261:11124-11130]. A gene from Brazil nut for a 2S albumin containing 18% methionine and 8% cysteine has been isolated [Altenbach et al., (1987) Plant Mol. Biol. 8:239-250]. Finally, a gene coding for a 10 kD seed prolamin containing 19% methionine and 10% cysteine has been isolated from rice [Masumura et al., (1989) Plant Mol. Biol. 12:123-130].
Plant breeders have long been interested in using naturally-occuring variations to improve protein quality and quantity in crop plants [Deutscher, (1978) Adv. Exp. Medicine and Biology 105:281-300]. Maize lines containing higher levels of lysine (70% increase) and tryptophan (100% increase) have been identified [Mertz, (1964) Science 145:279 and Nelson, (1965) Science 150:1469-70]. However, these lines which incorporate a mutant gene, opaque-2, exhibit poor agronomic qualities (increased susceptibility to disease and pests, 8-14% reduction in yield, low kernel weight, slower drying, lower dry milling yield of flaking grits, and increased storage problems) and are not commercially useful [Deutscher, (1978) Adv. Exp. Medicine and Biology 105:281-300]. Further breeding to improve the agronomics of opaque-2 lines is complicated because several modifier genes are involved which have complex inheritence patterns [Vasal, S. K. (1974) Symposium Proceedings. Worldwide Maize Improvement in the 70's and the Role for CIMMYT. Centro Internacional de MeJoramiento de Maiz y Trigo. El Batan, Mexico]. In spite of the difficulties, a few researchers have continued to work with opaque-2 mutants. Quality Protein Maize (QPM), bred at CIMMYT using the opaque-2 and sugary-2 genes and associated modifiers, has a hard endosperm and enriched levels of lysine and tryptophan in the kernals [Vasal et al., Proceedings of the 3rd Seed Protein Symposium, Gatersleben, Aug. 31-Sep. 2, (1983)].
However, the gene pools represented in the QPM lines are tropical and subtropical and they are only available as open pollinated types (hybrids are mainly used in the United States) [National Research Council Report (1988) Quality Protein Maize. National Academy Press, Washington, D.C.]. QPM is genetically complex and the existing lines are not easily adapted to the dent germplasm used in the United States. These factors prevent the adoption of QPM by U.S. corn breeders.
Efforts to improve the sulfur amino acid content of crops through traditional plant breeding have met with limited success on the laboratory scale and no success on the commercial scale. A mutant corn line with an elevated whole-kernel methionine concentration was isolated from corn cells grown in culture by selecting for growth in the presence of inhibitory concentrations of lysine plus threonine [Phillips et al., (1985) Cereal Chem. 62:213-218]. However, agronomically-acceptable cultivars have not yet been derived from this line.
Traditional breeding efforts designed to increase the level of methionine in soybean protein have been limited compared to efforts to increase overall protein quantity in soybeans [Burton, 1984 World Soybean Research Conference III Proceedings, p. 361-368 (Aug. 12-Aug. 17, 1984)]. Soybean cell lines with increased intracellular concentrations of methionine were isolated by selection for growth in the presence of ethionine, a nonmetabolizable methionine analog, Madison et al., (1988) Plant Cell Reports 7: 472-476, but plants were not regenerated from these lines.
Recombinant DNA technology offers the potential for altering the amino acid composition of crop plants. Particularly useful technologies are: (a) methods for the molecular cloning and in vitro manipulation of genes [see Sambrook et al., ( 1989 ) Molecular Cloning: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press], (b) introduction of genes via transformation into agriculturally-important crop plants such as soybean [Chee et al., (1989) Plant Physiol. 91: 1212-1218; Christou et al., (1989) Proc. Nat. Acad. Sci U.S.A. 86:7500-7504; Hinchee et al., (1989) Biotechnology 6:915-922; EPO publication 0301 749 A2], rapeseed [De Block et al., (1989) Plant Physiol. 91:694-701], and corn [Gordon-Kamm et al., (1990) Plant Cell 2:603-618; Fromm et al., (1990) Biotechnology 8:833-839], and (c) seed-specific expression of introduced genes in transgenic plants [see Goldberg et al., (1989) Cell 56:149-160); Thompson et al., (1989) BioEssays 10:108-113]. In order to use these technologies to develop crop plants with increased lysine and sulfur amino acid content, it is essential to obtain or develop commercially-important gene products enriched in the appropriate amino acids.
Expression of seed storage protein genes in transgenic plants have been reported in the model plant systems, tobacco or petunia, because it has only recently become possible to transform agriculturally-important crop plants such as corn and soybean. In general, dicot seed storage protein genes were expressed in a seed-specific manner in transformed dicot plants. Furthermore, both temporal and spatial control of gene expression was maintained. The transgenic protein products have, in some cases, been shown to be correctly processed and assembled into appropriate multimeric forms. [Beachy et al., (1985) EMBO J. 4:3047-3053; Sengupta-Gopalan et al., (1985) Proc. Natl. Acad. Sci. USA 82:3320-3324; Barker et al., (1988) Proc. Natl. Acad. Sci. USA 85:458-462; Ellis et al., (1988) Plant Mol. Biol. 10:203-214; Naito et al., (1988) Plant Mol. Biol. 11:109-123; Hoffman et al., (1988) Plant Mol. Biol. 11:717-729; Altenbach et al., (1989) Plant Mol. Biol. 13: 513-522].
Storage proteins are usually targetted to subcellular locales by the processing of N-terminal signal peptides or carboxy-terminal sequences such as SEKDEL [Bednareket al., (1990) The Plant Cell, 2: 1145-1155. Munro et al., (1987) Cell 48:899-907; Pelham, (1988) EMBO J. 7:913-918; Pelham et al., (1988) EMBO J. 1757-1762; Inohara et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:3564-3568; Hesse et al., (1989) EMBO J. 8:2453-2461]. It may prove necessary to create chimeric genes incorporating these signals for proper localization and stability of synthetic storage proteins.
Expression of seed storage protein genes in the leaves of plants is accomplished by replacing the regulatory signals that function in the seed with signals that function in the leaf [Lawton et al., (1987) Plant Mol. Biol. 9:315-324; Schernthaner et al., (1988) EMBO J. 7:1249-1255]. Monocot seed storage protein genes were expressed at very low levels [Schernthaner et al., (1988) EMBO J. 7:1249-1255] and, in one case, expressed in a non-seed-specific manner in transformed dicot plants [Ueng et. al., (1988) Plant Physiol. 86:1281-1285]. Replacement of the monocot regulatory regions (promoter and transcription terminator) with dicot seed-specific regulatory regions resulted in low level seed-specific expression of the protein in one case [Williamson et. al., (1988) Plant Physiol. 88:1002-1007]. In another case, high-level seed-specific expression of the monocot protein was found and the signal sequence of the monocot precursor was also correctly processed [Hoffman et al., (1987) EMBO J. 6:3213-3221].
In order to increase the lysine and sulfur amino acid contents of seeds, it is essential to obtain a gene or genes coding for a protein rich in lysine and the sulfur-containing amino acids methionine and cysteine. Methionine is preferable to cysteine because methionine can be converted to cysteine by most animals, while cysteine cannot be converted to methionine. It is desirable that the introduced protein be compatible with the target crop plant. It is desirable to select the gene to maximize lysine and sulfur amino acid content thereby minimizing the level of expression required in the plant to satisfy end-user needs. For this reason, those skilled in the art have not restricted themselves to natural genes and their polypeptide products.
Jaynes et al. worked with synthetic polypeptides which have elevated levels of lysine, methionine, tryptophan, threonine, and isoleucine as compared to known proteins [WO 89/04371]. These synthetic genes were formed by random ligation of mixtures of small oligodeoxy-nucleotides containing a high proportion (25-60%) of codons for essential amino acids. The proteins so formed are heterogeneous and do not fold to defined structures. Limited expression (0.02-0.35% of total plant protein) has been demonstrated in potato [Yang et al., (1989) Plant Science, 64: 99-111].
Others have modified natural proteins by addition or replacement of amino acids to increase the lysine or methionine content [DeClercq et al., EP 0 318 341 A1]. DeClercq et al. have Shown that it is possible to express modified storage protein genes in tobacco, Arabidopsis, and Brassica plants. However, their work gives little guidance regarding the design of the sequences rich in appropriate amino acids which are to be inserted into a target gene product. They observe only that the stability of the molecule should not be influenced and that long stretches of methionines should be interrupted by amino acids which break helical structures. Furthermore, the specific polypeptide sequences inserted into the target gene were not designed to adopt uniquely defined stable structures.
The importance of gene product stability in the seed dictates the need for a polypeptide of defined structure, while the need to complement existing amino acid composition and to satisfy end-user requirements emphasizes the importance of a flexible system which can accomodate variations in composition without sacrificing final gene product stability.